The unfolded protein response (UPR) in cultured cells expressing either wild-type or mutant hepatitis B e antigen (HBeAG) of the hepaptitis B virus(HBV)
Date
2014-02-18
Authors
Bhoola, Nimisha Harshadrai
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Abstract
Hepatitis B virus (HBV) is hyperendemic to southern Africa, the Asia-Pacific region, and
the Amazon Basin. HBV causes both acute, and chronic infection of the liver that result in
a wide spectrum of liver diseases ranging from acute mild subclinical infection, and an
asymptomatic carrier state (ASC) to severe clinical manifestations, including, severe acute
and, chronic hepatitis, which can progress to cirrhosis, and the development of
hepatocellular carcinoma (HCC). Several viral factors have been implicated in the
activation, and inhibition of apoptosis. The development, and progression of this wide
spectrum of liver diseases are associated with an unregulated increase or decrease in
hepatocyte apoptosis as well as a loss of balance between cell proliferation, and apoptosis.
In southern Africa, genotype A of HBV is the predominant genotype, with subgenotype A1
prevailing. Individuals infected with subgenotype A1 have several unique characteristics,
including relatively lower levels of HBV DNA, and early seroconversion of hepatitis B e
antigen (HBeAg) to antibodies against HBeAg during the course of HBV infection.
Infection with this subgenotype is associated with rapid disease progression, and high
frequency of HCC development. Moreover, patients infected with subgenotype A1 had
increased levels of apoptosis when compared to the other subgenotypes. The G1862T
mutation occurs most frequently in subgenotype A1. In the clinical setting, South African
HCC patients infected with G1862T subgenotype A1 strains had higher levels of apoptosis
while ASCs patients infected with G1862T subgenotype A1 strains had lower levels of
apoptosis, when compared to those infected with wild-type. To date, G1862T has been
functionally characterized in subgenotype A2, genotype B and in a genotype D HBV
genome containing a subgenotype A1 precore (PreC) region.
The objectives of our study were to construct 1.28 mer replication competent clones
containing an endogenous HBV promoter for wild-type subgenotypes A1, A2, and D3 as
well as mutant G1862T subgenotype A1, and to functionally characterize these strains in
tissue culture. Transfection of Huh7 cells was used to follow the viral replication,
expression of HBeAg, activation of the unfolded protein response (UPR), and subsequent
apoptosis.
The strategy used for the generation of these replication competent clones had several
advantages. Very few PCR errors were introduced, and carry-over of enzymes, nonspecific
products, and reaction reagents downstream was prevented. The clones contained
endogenous HBV promoters, and enhancers, and were generated from a single complete
genome of HBV. These replication competent clones may be used in future studies for the
establishment of stable cell lines that constitutively express HBV proteins without the need
for further manipulation. This strategy can be used for the generation of replication
competent clones belonging to genotypes A to D, and G, and with a few minor
modifications, for genotypes E, F, and H.
Using the newly generated clones, their replication competence was demonstrated using
transfection of Huh7 cells. Even in the absence of an exogenous promoter, these clones
were able to support the expression of intracellular, and extracellular HBV DNA at levels
of 108 to 109 genome copies/ml. HBcAg, HBeAg, and HBsAg were expressed for a period
of five days, and the order of expression was similar to that seen during acute HBV
infection.
Comparison of transfection with a replication competent clone containing an exogenous
HBV promoter demonstrated higher expression of HBV DNA, and proteins, as well as an
earlier expression, and accumulation of HBeAg in the endoplasmic reticulum (ER) relative
to the clone containing an endogenous HBV promoter. This initial increased accumulation
of HBeAg in the ER did not affect the level of activation of the UPR, but led to an
increased level of total cell death as a consequence of necrosis.
When comparing the different subgenotypes following transfection into Huh7 cells,
subgenotype D3 replicated at a lower level, as measured by HBsAg, and HBV DNA levels,
with HBeAg passing through the secretory pathway earlier, when compared to cells
transfected with genotype A. There was no difference in the intracellular, and extracellular
HBsAg between cells transfected with either subgenotype A1 or A2. However, cells
transfected with subgenotype A1 had higher levels of intracellular replicative
intermediates, HBeAg, and HBcAg, and lower extracellular expression of HBeAg from
days 1 to 3, when compared to cells transfected with subgenotype A2. The intracellular
retention of the PreC/ core (C) precursor protein in cells transfected with subgenotype A1
was clearly demonstrated by its lower expression in the secretory pathway, and its higher
co-localization in the nucleus, using indirect immunofluorescence. This intracellular
retention led to greater ER stress, and an earlier, and prolonged activation of the UPR. This
correlated well with the higher PERK, ATF6, and IRE1/XBP1 activity seen on days 3 than
on day 5. These findings suggest that the prolonged activation of the UPR in cells
transfected with subgenotype A1 led to increased apoptosis, and subsequent induction of
liver damage, and may therefore, be a contributing factor to the higher hepatocarcinogenic
potential of subgenotype A1.
Our study demonstrated that G1862T reduced replication, and led to the initial temporal
retardation of intracellular core-particle-associated HBV DNA. Although, G1862T did not
affect HBsAg expression, it led to a decreased expression of HBcAg, and HBeAg. The
decreased expression of extracellular HBeAg was probably as a result of decreased
cleavage efficiency by the signal peptide, which consequently led to the retardation of the
PreC/C precursor protein in the ER, and ER-Golgi intermediate compartment (ERGIC),
and its decreased expression in the nucleus. This retardation, and accumulation led to the
earlier activation of all three UPR pathways, but not to increased apoptosis. Therefore, it is
evident that G1862T does not completely abolish HBeAg expression, but affects the rate of
HBeAg maturation, and its expression through the secretory pathway. These findings
suggest that in response to the accumulation of HBeAg in the ER, the UPR was activated
resulting in the alteration of the capacity to overcome this stress, consequently leading to a
new homeostasis of the ER being reached. The capacity of the ER is increased, with no
further activation of the UPR and apoptosis, which facilitates maturation of HBeAg.
In conclusion, our study for the first time demonstrated that there are a number of factors
that influence the expression of proteins in HBV transfection studies including the type of
transcriptional promoter, the different genotypes/subgenotypes of HBV, the use of protein
expressing as opposed to replication competent clones, and the presence, and absence of
mutations, such as the G1862T. Therefore, when comparing the outcomes of various
experiments these factors should be taken into consideration, and the results interpreted
with caution, because experiments may not be strictly speaking comparable. Importantly,
replication competent clones were generated from strains circulating in southern Africa.
The generation of these clones is an important step in further functional characterization of
African strains of HBV, and their comparison to strains circulating other geographical
regions of the world. These strains, in particular, subgenotype A1 can develop unique
mutations, such as the G1862T, which we demonstrated can influence the expression of
HBeAg, in a way that it can possibly account for the higher hepatocarcinogenic potential
of subgenotype A1.